ES2564087T3 - Method to measure calcineurin activity - Google Patents
Method to measure calcineurin activity Download PDFInfo
- Publication number
- ES2564087T3 ES2564087T3 ES13703836.0T ES13703836T ES2564087T3 ES 2564087 T3 ES2564087 T3 ES 2564087T3 ES 13703836 T ES13703836 T ES 13703836T ES 2564087 T3 ES2564087 T3 ES 2564087T3
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- Prior art keywords
- calcineurin
- substrate
- activity
- biological sample
- inhibitor
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- 229940085991 phosphate ion Drugs 0.000 description 1
- ZLKNTGQAQQSIFV-HQAKDUOCSA-N pkip Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)[C@@H](C)O)[C@@H](C)CC)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 ZLKNTGQAQQSIFV-HQAKDUOCSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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Abstract
Un método para medir la actividad de calcineurina en una muestra biológica que comprende las etapas de: - proporcionar una mezcla de reacción que comprende un sustrato, al menos un inhibidor de fosfatasa y al menos un inhibidor de quinasa; - incubar dicha muestra biológica con la mezcla de reacción en condiciones adecuadas para la actividad de calcineurina; - medir la cantidad de sustrato desfosforilado.A method for measuring calcineurin activity in a biological sample comprising the steps of: - providing a reaction mixture comprising a substrate, at least one phosphatase inhibitor and at least one kinase inhibitor; - incubating said biological sample with the reaction mixture under conditions suitable for calcineurin activity; - measuring the amount of dephosphorylated substrate.
Description
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DESCRIPCIONDESCRIPTION
Metodo para medir la actividad de calcineurina Sector de la tecnicaMethod for measuring calcineurin activity Technical sector
La presente invencion se refiere a un metodo para medir la actividad de calcineurina en una muestra biologica. Estado de la tecnicaThe present invention relates to a method for measuring calcineurin activity in a biological sample. State of the art
El patron para la prevencion de rechazo en pacientes de trasplante consiste en regfmenes inmunosupresores que incluyen un inhibidor de la calcineurina tal como ciclosporina (CsA) o tacrolimus (TAC). Sin embargo, despues del trasplante es diffcil conseguir el equilibrio optimo entre una inmunosupresion demasiado fuerte o una demasiado debil. Una inmunosupresion inadecuada puede provocar un rechazo al trasplante y, por otro lado, una inmunosupresion excesiva facilita el desarrollo de complicaciones graves tales como infecciones o trastornos linfoproliferativos. La practica habitual consiste, para los medicos, en establecer el tratamiento con dosis fijas, a continuacion en el ajuste de las dosis de farmaco de acuerdo con sus niveles en sangre o, incluso de forma mas frecuente, de acuerdo con la aparicion bien de un rechazo agudo o bien de un suceso de farmaco adverso. Desafortunadamente, esta estrategia se asocia con un fallo frecuente como se ilustra con el hecho de que de un 30 a un 50 % de los pacientes desarrollan rechazo agudo despues del trasplante.The pattern for the prevention of rejection in transplant patients consists of immunosuppressive regimes that include a calcineurin inhibitor such as cyclosporine (CsA) or tacrolimus (TAC). However, after transplantation it is difficult to achieve the optimal balance between an immunosuppression too strong or too weak. Inadequate immunosuppression can cause transplant rejection and, on the other hand, excessive immunosuppression facilitates the development of serious complications such as infections or lymphoproliferative disorders. The usual practice is, for doctors, to establish the treatment with fixed doses, then in adjusting the drug doses according to their blood levels or, even more frequently, according to the appearance of a Acute rejection or of an adverse drug event. Unfortunately, this strategy is associated with a frequent failure as illustrated by the fact that 30 to 50% of patients develop acute rejection after transplantation.
Un enfoque farmacodinamico, basado en la medida del efecto de los farmacos en sus dianas celulares, se ha desarrollado con el objetivo de reducir la incidencia y gravedad de rechazo agudo. Este enfoque farmacodinamico se basa en la medida de la actividad de la calcineurina (CN), una fosfatasa dependiente de calcio y calmodulina. La CN es parte de la familia de la enzima serina/treonina fosfatasa que incluye PP1, PP2A, PP2B o calcineurina y PP2C (Ingebritsen y Cohen, 1983). Los ensayos actuales sobre calcineurina se realizan generalmente en extractos celulares obtenidos a partir de celulas mononucleares sangufneas (PBMC, celulas mononucleares de sangre periferica) de pacientes de trasplante (Fruman et al., 1996; Sanquer et al., 2004; Fukudo et al., 2005). Estos ensayos se basan en la desfosforilacion de un sustrato de calcineurina fosforilado en presencia de calcio y calmodulina queA pharmacodynamic approach, based on the measure of the effect of drugs on their cell targets, has been developed with the aim of reducing the incidence and severity of acute rejection. This pharmacodynamic approach is based on the measurement of the activity of calcineurin (CN), a calcium-dependent phosphatase and calmodulin. CN is part of the serine / threonine phosphatase enzyme family that includes PP1, PP2A, PP2B or calcineurin and PP2C (Ingebritsen and Cohen, 1983). Current trials on calcineurin are generally performed on cell extracts obtained from blood mononuclear cells (PBMC, peripheral blood mononuclear cells) from transplant patients (Fruman et al., 1996; Sanquer et al., 2004; Fukudo et al. , 2005). These assays are based on the dephosphorylation of a phosphorylated calcineurin substrate in the presence of calcium and calmodulin that
son esenciales para la actividad de la enzima (Pallen y Wang, 1983). La adicion de acido okadaico a unathey are essential for enzyme activity (Pallen and Wang, 1983). The addition of okadaic acid to a
concentracion de 500 nM (que inhibe todas las fosfatasas excepto CN) evitar la reactividad cruzada potencial para el sustrato fosforilado.500 nM concentration (which inhibits all phosphatases except CN) avoid potential cross-reactivity for the phosphorylated substrate.
Un sustrato especialmente preferente es el peptido fosforilado RII (P-RII), derivado de la subunidad reguladora de la protefna quinasa A (PKA, Donella-Deana et al., 1994). Este peptido se caracteriza por la siguiente secuencia: DLDVPIPGRFDRRVpSVAAE (SEC ID N°: 1).An especially preferred substrate is the phosphorylated peptide RII (P-RII), derived from the regulatory subunit of protein kinase A (PKA, Donella-Deana et al., 1994). This peptide is characterized by the following sequence: DLDVPIPGRFDRRVpSVAAE (SEQ ID NO: 1).
En los diferentes ensayos descritos hasta ahora, el peptido P-RII se puede etiquetar o no, y el etiquetado se realiza bien con un radioelemento (32P) o un fluoroforo.In the different tests described so far, the P-RII peptide can be labeled or not, and the labeling is done either with a radioelement (32P) or a fluorophore.
La mayorfa de los metodos usados para medir la actividad de la CN fosfatasa se basan en la deteccion del fosfato libre liberado durante la reaccion enzimatica. Estos metodos usan medidas de radiometrfa en el caso del peptido P- RII etiquetado con 32P (Fruman et al., 1996; Koefoed-Nielsen et al., 2004). Ellos usan medidas deMost of the methods used to measure the activity of CN phosphatase are based on the detection of free phosphate released during the enzymatic reaction. These methods use radiometry measurements in the case of the P-RII peptide labeled with 32P (Fruman et al., 1996; Koefoed-Nielsen et al., 2004). They use measures of
espectrofotometrfa en el caso del peptido R-RII sin etiquetar. Este ultimo requiere la puesta en practica de unaspectrophotometry in the case of the unlabeled R-RII peptide. The latter requires the implementation of a
segunda reaccion con verde de malaquita para colorear el fosfato inorganico libre (Sellar et al., 2006).second reaction with malachite green to color free inorganic phosphate (Sellar et al., 2006).
Se han desarrollado muchos metodos especfficos basados en la medida directa de la formacion del peptido RII desfosforilado (DP-RII) con CN. Estos metodos se basaban originalmente en cromatograffa lfquida acoplada a deteccion de UV (Enz et al., 1994; Sanquer et al., 2004). Estos metodos se desarrollaron para el peptido P-RII sin etiquetar. Mas recientemente, se describio un metodo de fluorimetrfa para medir la actividad de CN mediante el uso del peptido P-RII etiquetado con un fluoroforo y separacion del producto de peptido desfosforilado con oxido de titanio (Roberts et al., 2008).Many specific methods have been developed based on the direct measurement of the formation of the dephosphorylated RII peptide (DP-RII) with CN. These methods were originally based on liquid chromatography coupled to UV detection (Enz et al., 1994; Sanquer et al., 2004). These methods were developed for the unlabeled P-RII peptide. More recently, a fluorimetry method for measuring CN activity was described by using a fluorophore-labeled P-RII peptide and separating the dephosphorylated peptide product with titanium oxide (Roberts et al., 2008).
El documento WO2011/085176 describe un metodo basado en FRET para determinar la actividad de fosfatasa actividad de la calcineurina.WO2011 / 085176 describes a method based on FRET for determining the activity of calcineurin phosphatase activity.
Objeto de la invencionObject of the invention
La invencion surge de la observacion de que algunas muestras biologicas, tales como extractos de celulas mononucleares de sangre periferica, contienen una actividad de quinasa que puede competir con la actividad de fosfatasa de la calcineurina. Esta actividad de quinasa, que nunca antes se informo en los metodos para medir la actividad de calcineurina de la tecnica anterior, se deberfa inhibir para obtener resultados mas especfficos.The invention arises from the observation that some biological samples, such as extracts of peripheral blood mononuclear cells, contain a kinase activity that can compete with calcineurin phosphatase activity. This kinase activity, which was never reported before in the methods for measuring the calcineurin activity of the prior art, should be inhibited to obtain more specific results.
De hecho, los inventores han observado que la especificidad del ensayo de actividad de calcineurina puede aumentar de forma significativa mediante la adicion de al menos un inhibidor de quinasa a la mezcla de reaccion.In fact, the inventors have observed that the specificity of the calcineurin activity assay can be significantly increased by adding at least one kinase inhibitor to the reaction mixture.
Por lo tanto, la presente invencion se refiere a un metodo para medir la actividad de calcineurina en una muestra biologica que comprende las etapas de:Therefore, the present invention relates to a method for measuring calcineurin activity in a biological sample comprising the steps of:
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- proporcionar una mezcla de reaccion que comprende un sustrato, al menos un inhibidor de fosfatasa y al menos un inhibidor de quinasa;- providing a reaction mixture comprising a substrate, at least one phosphatase inhibitor and at least one kinase inhibitor;
- incubar dicha muestra biologica con la mezcla de reaccion en condiciones adecuadas para la actividad de calcineurina;- incubating said biological sample with the reaction mixture under conditions suitable for calcineurin activity;
- medir la cantidad de sustrato desfosforilado.- measure the amount of dephosphorylated substrate.
La presente invencion tambien se refiere a un kit para medir la actividad de calcineurina en una muestra biologica que comprende un sustrato, al menos un inhibidor de fosfatasa y al menos un inhibidor de quinasa.The present invention also relates to a kit for measuring calcineurin activity in a biological sample comprising a substrate, at least one phosphatase inhibitor and at least one kinase inhibitor.
Otro aspecto de la invencion se refiere al uso de inhibidor de quinasa en un metodo para medir la actividad de calcineurina.Another aspect of the invention relates to the use of kinase inhibitor in a method for measuring calcineurin activity.
En otro aspecto mas, la invencion se refiere a un metodo para predecir el resultado terapeutico de un paciente de trasplante que comprende medir la actividad de calcineurina en una muestra biologica obtenida de dicho paciente como se ha descrito anteriormente.In yet another aspect, the invention relates to a method for predicting the therapeutic outcome of a transplant patient that comprises measuring calcineurin activity in a biological sample obtained from said patient as described above.
Descripcion detallada de la invencionDetailed description of the invention
La presente invencion se refiere a un metodo para medir la actividad de calcineurina en una muestra biologica que comprende las etapas de:The present invention relates to a method for measuring calcineurin activity in a biological sample comprising the steps of:
- proporcionar una mezcla de reaccion que comprende un sustrato, al menos un inhibidor de fosfatasa y al menos un inhibidor de quinasa;- providing a reaction mixture comprising a substrate, at least one phosphatase inhibitor and at least one kinase inhibitor;
- incubar dicha muestra biologica con la mezcla de reaccion en condiciones adecuadas para la actividad de calcineurina;- incubating said biological sample with the reaction mixture under conditions suitable for calcineurin activity;
- medir la cantidad de sustrato desfosforilado.- measure the amount of dephosphorylated substrate.
Como se usa en el presente documento el termino "calcineurina", se usa indistintamente con "CaN", "fosfatasa 2B" o "PP-2B", y se refiere a la protefna fosfatasa de Serina /Treonina dependiente de Ca2+/calmodulina. Esta protefna cataliza la retirada de un grupo fosfato de su sustrato por hidrolisis de monoesteres de acido fosforico en un ion fosfato y una molecula con un grupo hidroxilo libre. De forma mas especffica, la calcineurina retira un grupo fosfato de un resto de serina fosforilada (fosfo-serina, o "pS") o un resto de treonina fosforilada (fosfo-treonina, o "pT").As the term "calcineurin" is used herein, it is used interchangeably with "CaN", "phosphatase 2B" or "PP-2B", and refers to the Ca2 + / calmodulin-dependent serine / Threonine-dependent phosphatase protein. This protein catalyzes the removal of a phosphate group from its substrate by hydrolysis of phosphoric acid monoesters in a phosphate ion and a molecule with a free hydroxyl group. More specifically, calcineurin removes a phosphate group from a phosphorylated serine moiety (phospho-serine, or "pS") or a phosphorylated threonine moiety (phospho-threonine, or "pT").
La calcineurina es un heterodfmero que consiste en una subunidad A catalftica (57-61 kDa) y una subunidad B reguladora (19 kDa). La subunidad A catalftica esta formada por cuatro dominios funcionales: el nucleo catalftico con homologfa de secuencias para PP-1 y PP-2A; sitios de union tanto para calmodulina como para la subunidad reguladora de CaN, y dominio autoinhibidor C-terminal.Calcineurin is a heterodimer consisting of a catalytic A subunit (57-61 kDa) and a regulatory B subunit (19 kDa). The catalytic subunit A is formed by four functional domains: the catalytic nucleus with sequence homology for PP-1 and PP-2A; binding sites for both calmodulin and the regulatory subunit of CaN, and C-terminal autoinhibitor domain.
Como se usa en el presente documento, el termino "sustrato" o "sustrato de calcineurina" se refiere a una fosfoprotefna o fosfopeptido que se puede hidrolizar por calcineurina. Por lo tanto, se refiere a una fosfoprotefna o fosfopeptido que tiene al menos un resto de fosfoserina y/o fosfotreonina.As used herein, the term "substrate" or "calcineurin substrate" refers to a phosphoprotephne or phosphopeptide that can be hydrolyzed by calcineurin. Therefore, it refers to a phosphoprotephne or phosphopeptide having at least one phosphoserine and / or phosphotreonin moiety.
Se prefiere que los restos de aminoacido que se describen en el presente documento esten en la forma isomerica "L". Sin embargo, algunos restos en la forma isomerica "D" se pueden sustituir para cualquier resto de L-aminoacido, siempre y cuando se mantenga la capacidad del sustrato para ser hidrolizado por la calcineurina. NH3 se refiere al grupo amino libre presente en el extremo amino de un polipeptido. COOH se refiere al grupo carboxi libre presente en extremo carboxi de un polipeptido.It is preferred that the amino acid residues described herein be in the "L" isomeric form. However, some residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the ability of the substrate to be hydrolyzed by calcineurin is maintained. NH3 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
De acuerdo con la nomenclatura convencional de polipeptidos, J. Biol. Chem., 243: 3552-59 (1969), en la siguiente Tabla de Correspondencias se muestran algunas abreviaturas para restos de aminoacido:According to the conventional polypeptide nomenclature, J. Biol. Chem., 243: 3552-59 (1969), some abbreviations for amino acid residues are shown in the following Correspondence Table:
- Codigo de una letra One letter code
- Codigo de tres letras Aminoacido Three-letter code Amino acid
- Y Y
- Tyr tirosina Tyr Tyrosine
- G G
- Gly glicina Glycine glycine
- F F
- Phe fenilalanina Phe phenylalanine
- M M
- Met metionina Met Methionine
- A TO
- Ala alanina Alanine wing
- S S
- Ser serina Be serine
- I I
- Ile isoleucina Ile isoleucine
- L L
- Leu leucina Leu Leucine
- T T
- Thr treonina Thr threonine
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- Codigo de una letra One letter code
- Codigo de tres letras Aminoacido Three-letter code Amino acid
- V V
- Val valina Val Valine
- P P
- Pro prolina Pro proline
- K K
- Lys lisina Lys lysine
- H H
- His histidina His histidine
- Q Q
- Gln glutamina Gln glutamine
- E AND
- Glu acido glutamico Glutamic acid
- W W
- Trp triptofano Tryptophan Trp
- R R
- Arg arginina Arg arginine
- D D
- Asp acido aspartico Asp aspartic acid
- N N
- Asn asparagina Asnragine Asn
- C C
- Cys cistefna Cys cystefna
En el presente documento, todas las secuencias de restos de aminoacido ase representan con formulas cuya orientacion a la izquierda y la derecha esta en la direccion convencional del extremo amino-terminal con respecto al extremo carboxi-terminal.Here, all amino acid residue sequences are represented by formulas whose orientation to the left and right is in the conventional direction of the amino-terminal end with respect to the carboxy-terminal end.
El termino "pX", o "fosfoX", como se usa en el presente documento, se refiere a un resto de aminoacido X fosforilado. Por lo general, el sustrato de acuerdo con la invencion comprende al menos un resto de fosfoserina o fosfotreonina.The term "pX", or "fosfoX", as used herein, refers to a phosphorylated amino acid residue X. Generally, the substrate according to the invention comprises at least one phosphoserine or phosphotreonin moiety.
Algunos estratos preferentes de acuerdo con la invencion son sustratos especfficos de calcineurina. En otros terminos, el sustrato de acuerdo con la invencion por lo general se reconoce y se hidroliza con calcineurina de forma mas eficaz que con otros tipos de fosfatasas.Some preferred strata according to the invention are specific calcineurin substrates. In other terms, the substrate according to the invention is generally recognized and hydrolyzed with calcineurin more efficiently than with other types of phosphatases.
Por lo general, el sustrato de acuerdo con la invencion se puede seleccionar entre el grupo que consiste en los fosfopeptidos que se describen en la Tabla I de Donella-Deana et al. (Donella-Deana et al., 1994, Eur J Biochem 219, 109-117).Generally, the substrate according to the invention can be selected from the group consisting of the phosphopeptides described in Table I of Donella-Deana et al. (Donella-Deana et al., 1994, Eur J Biochem 219, 109-117).
Por lo general, un sustrato preferente es un fragmento de la subunidad reguladora de la protefna quinasa de tipo II dependiente de cAMP (RII). En una realizacion mas preferente, el sustrato es DLDVPIPGRFDRRVpSVAAE (SEC ID N°:1).Generally, a preferred substrate is a fragment of the regulatory subunit of cAMP-dependent type II protein kinase (RII). In a more preferred embodiment, the substrate is DLDVPIPGRFDRRVpSVAAE (SEQ ID NO: 1).
El sustrato de la invencion se puede obtener con cualquier metodo adecuado en la tecnica: puede ser una fosfoprotefna recombinante o fragmento de la misma, una fosfoprotefna purificada o fragmento de la misma, o un fosfopeptido sintetico.The substrate of the invention can be obtained by any suitable method in the art: it can be a recombinant phosphoprotephine or fragment thereof, a purified phosphoprotephine or fragment thereof, or a synthetic phosphopeptide.
En una realizacion de la intervencion, el sustrato es un fosfopeptido sintetico.In one embodiment of the intervention, the substrate is a synthetic phosphopeptide.
El sustrato de acuerdo con la invencion puede estar etiquetado, o sin etiquetar.The substrate according to the invention may be labeled, or unlabeled.
La deteccion del producto de reaccion de desfosforilacion se realizara a continuacion con diferentes medios, de acuerdo con el tipo de sustrato que se use (vease a continuacion).The detection of the dephosphorylation reaction product will then be carried out with different means, according to the type of substrate used (see below).
Como se usa en el presente documento, la expresion "condiciones adecuadas para la actividad de calcineurina" tiene un significado general en la tecnica y se refiere a las condiciones fisicoqufmicas globales en que se produce la reaccion de desfosforilacion catalizada por calcineurina.As used herein, the expression "conditions suitable for calcineurin activity" has a general meaning in the art and refers to the overall physicochemical conditions in which the calcineurin catalyzed dephosphorylation reaction occurs.
De acuerdo con una realizacion, la reaccion se realiza a una temperatura comprendida entre 20 °C y 40 °C, preferentemente entre 25 °C y 38 °C, incluso mas preferentemente a aproximadamente 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C o 37 °C.According to one embodiment, the reaction is carried out at a temperature between 20 ° C and 40 ° C, preferably between 25 ° C and 38 ° C, even more preferably at about 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C or 37 ° C.
De acuerdo con una realizacion, la reaccion se realiza durante un periodo de tiempo comprendido entre 5 minutos y 60 minutos, preferentemente entre 10 y 45 minutos, entre 15 y 40 minutos, incluso mas preferentemente aproximadamente 30 minutos.According to one embodiment, the reaction is carried out for a period of time between 5 minutes and 60 minutes, preferably between 10 and 45 minutes, between 15 and 40 minutes, even more preferably about 30 minutes.
En una realizacion de la invencion, la reaccion se realiza por incubacion de la muestra biologica y la mezcla de reaccion a 37 °C durante 30 minutos.In one embodiment of the invention, the reaction is carried out by incubation of the biological sample and the reaction mixture at 37 ° C for 30 minutes.
Por lo general, la reaccion se puede detener despues de un periodo tiempo determinado previamente mediante colocacion del tubo de reaccion a +4 °C y adicion de acido tricloroacetico (TCA).In general, the reaction can be stopped after a period of time previously determined by placing the reaction tube at +4 ° C and adding trichloroacetic acid (TCA).
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De acuerdo con la invencion, la desfosforilacion del sustrato con calcineurina se realiza en una mezcla de reaccion. Como se usa en el presente documento, la expresion "mezcla de reaccion" o "mezcla de reaccion de ensayo" tiene su significado general en la tecnica. Y se refiere a la solucion en que se realiza la reaccion enzimatica a cuantificar. Por lo general, la mezcla de reaccion de ensayo de acuerdo con la invencion es una solucion acuosa que comprende todos los reactivos necesarios para que se produzca la reaccion enzimatica. Por lo general, la mezcla de reaccion comprende agua, un tampon de pH y sales.According to the invention, dephosphorylation of the substrate with calcineurin is performed in a reaction mixture. As used herein, the expression "reaction mixture" or "test reaction mixture" has its general meaning in the art. And it refers to the solution in which the enzymatic reaction to be quantified is performed. Generally, the test reaction mixture according to the invention is an aqueous solution comprising all the reagents necessary for the enzymatic reaction to occur. Generally, the reaction mixture comprises water, a pH buffer and salts.
Algunos tampones de pH adecuados de acuerdo con la invencion son tampones sin fosfato, tales como Tris-HCl.Some suitable pH buffers according to the invention are phosphate free buffers, such as Tris-HCl.
Por lo general, el pH de la mezcla de reaccion esta comprendido entre 6,0 y 8,0, preferentemente alrededor de 7,0.Generally, the pH of the reaction mixture is between 6.0 and 8.0, preferably around 7.0.
En una realizacion, el tampon de reaccion comprende Ca2+ y calmodulina. De hecho, la actividad de fosfatasa de la calcineurina nativa depende de la presencia de Ca2+ y calmodulina.In one embodiment, the reaction buffer comprises Ca2 + and calmodulin. In fact, the phosphatase activity of native calcineurin depends on the presence of Ca2 + and calmodulin.
Por lo general, la mezcla de reaccion de ensayo puede comprender una concentracion final de calmodulina comprendida entre 0,01 y 0,06 unidades, de forma preferente aproximadamente 0,03 unidades.Generally, the test reaction mixture may comprise a final concentration of calmodulin comprised between 0.01 and 0.06 units, preferably about 0.03 units.
Por lo general, la mezcla de reaccion de ensayo comprende una concentracion final de Ca2+ comprendida entre 30 pM y 90 pM, de forma preferente aproximadamente 50 pM.Generally, the test reaction mixture comprises a final concentration of Ca2 + comprised between 30 pM and 90 pM, preferably about 50 pM.
En una realizacion de la invencion, se anaden inhibidores de proteasa a la muestra biologica y/o a la mezcla de reaccion. De hecho, la presencia de tales inhibidores evita la degradacion de la calcineurina presente en la muestra biologica mediante proteasas endogenas. Algunos inhibidores de proteasa adecuados incluyen, pero no se limitan a PMSF, EDTA, EGTA, aprotinina, leupeptina, pepstatina y mezclas de los mismos.In one embodiment of the invention, protease inhibitors are added to the biological sample and / or the reaction mixture. In fact, the presence of such inhibitors prevents the degradation of calcineurin present in the biological sample by endogenous proteases. Some suitable protease inhibitors include, but are not limited to PMSF, EDTA, EGTA, aprotinin, leupeptin, pepstatin and mixtures thereof.
La reaccion de acuerdo con la invencion se realiza en presencia de al menos un inhibidor de fosfatasa. Como se usa en el presente documento, la expresion "inhibidor de fosfatasa" tiene su significado general en la tecnica. Se refiere a un compuesto que inhibe al menos una fosfatasa. Por lo general, el inhibidor de fosfatasa de acuerdo con la invencion no es un inhibidor de calcineurina a la concentracion seleccionada.The reaction according to the invention is carried out in the presence of at least one phosphatase inhibitor. As used herein, the term "phosphatase inhibitor" has its general meaning in the art. It refers to a compound that inhibits at least one phosphatase. Generally, the phosphatase inhibitor according to the invention is not a calcineurin inhibitor at the selected concentration.
Algunos inhibidores de fosfatasa adecuados de acuerdo con la invencion incluyen inhibidores especfficos de fosfatasa e inhibidores de amplio espectro.Some suitable phosphatase inhibitors according to the invention include specific phosphatase inhibitors and broad spectrum inhibitors.
Por lo general, el inhibidor de fosfatasa de acuerdo con la invencion es acido okadaico. El acido okadaico (9,10- Desepitio-9,10-dideshidroacantifolicina) es un potente inhibidor de la protefna fosfatasa 1 (PP-1; CI50 = 20-100 nM), protefna fosfatasa 2A (PP-2A; CI50 = 0,1-1 nM) y protefna fosfatasa 2B (PP-2B; CI50 > 5000 nM).Generally, the phosphatase inhibitor according to the invention is okadaic acid. Okadaic acid (9,10- Desepitio-9,10-dideshydroacantifolicin) is a potent inhibitor of protein phosphatase 1 (PP-1; IC50 = 20-100 nM), protein phosphatase 2A (PP-2A; IC50 = 0, 1-1 nM) and protein phosphatase 2B (PP-2B; IC50> 5000 nM).
Por lo general, el acido okadaico se usa a una concentracion final quedarfa de 100 a 1000 nM, de forma preferente aproximadamente 500 nM.Generally, okadaic acid is used at a final concentration of 100 to 1000 nM, preferably about 500 nM.
Otro inhibidor de fosfatasa adecuado es la microcistina-LR.Another suitable phosphatase inhibitor is microcystin-LR.
La reaccion de acuerdo con la presente invencion se realiza en presencia de al menos un inhibidor de quinasa. De hecho, los inventores han demostrado, por primera vez, que algunas muestras biologicas contenfan una actividad de quinasa que compite con la actividad de la calcineurina fosfatasa, lo que conducfa perdida de especificidad de los ensayos descritos anteriormente.The reaction according to the present invention is carried out in the presence of at least one kinase inhibitor. In fact, the inventors have demonstrated, for the first time, that some biological samples contained a kinase activity that competes with the activity of calcineurin phosphatase, which led to loss of specificity of the assays described above.
Por lo tanto, la realizacion del ensayo de actividad de calcineurina en presencia de al menos un inhibidor de quinasa conduce a un aumento de la especificidad del metodo.Therefore, performing the calcineurin activity assay in the presence of at least one kinase inhibitor leads to an increase in the specificity of the method.
Como se usa en el presente documento, la expresion "inhibidor de quinasa" se refiere a un compuesto que inhibe la actividad de al menos una quinasa.As used herein, the term "kinase inhibitor" refers to a compound that inhibits the activity of at least one kinase.
De acuerdo con una realizacion de la invencion, el inhibidor de quinasa es un inhibidor selectivo de la Protefna Quinasa A (PKA).According to an embodiment of the invention, the kinase inhibitor is a selective protein kinase A (PKA) inhibitor.
La seleccion de inhibidores de PKA apropiados entra dentro de la capacidad de la persona experta en la materia.The selection of appropriate PKA inhibitors falls within the capacity of the person skilled in the art.
Algunos inhibidores de PKA selectivos adecuados de acuerdo con la invencion pueden ser 1) analogos estructurales de cAMP, la familia de los Rp-cAMP, que son inhibidores competitivos del sitio de union a cAMP, 2) inhibidores endogenos de la actividad de PKA: el peptido inhibidor de la protefna quinasa (Murray, 2008) y 3) ARN de silenciamiento dirigido a al menos las dos isoformas (a y p) de la subunidad catalftica de PKA (Murray, 2008; Dumaz et al., 2003; Rudolph et al., 2007; Monagham et al., 2008). Estos compuestos incluyen, pero no se limitan a Rp- adenosin 3',5'-monofosforotioato cfclico (Wang et al., 1991); Rp-8-Bromoadenosin 3',5'-Monofosforotioato cfclico (Gjertsen et al., 1995); Rp-8-bromo-2'-O-monobutiriladenosin-3',5'-monofosforotioato cfclico (Ruiz-Velasco et al.,Some suitable selective PKA inhibitors according to the invention may be 1) structural analogs of cAMP, the family of Rp-cAMP, which are competitive inhibitors of the cAMP binding site, 2) endogenous inhibitors of PKA activity: Protein kinase inhibitor peptide (Murray, 2008) and 3) silencing RNA targeting at least the two isoforms (a and p) of the PKA catalytic subunit (Murray, 2008; Dumaz et al., 2003; Rudolph et al., 2007; Monagham et al., 2008). These compounds include, but are not limited to Rp-adenosine 3 ', 5'-cyclic phosphorothioate (Wang et al., 1991); Rp-8-Bromoadenosin 3 ', 5'-Cylphosphorothioate (Gjertsen et al., 1995); Rp-8-bromo-2'-O-monobutyryladenosin-3 ', 5'-cyclic monophosphorothioate (Ruiz-Velasco et al.,
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1998); Rp-8-cloroadenosin-3’,5’-monofosforotioato cfclico (Yokozaki et al., 1992); Rp-8-(4-clorofeniltio)adenosin-3’,5’- monofosforotioato cfclico (Weisskupf et al., 1994); Rp-8-hexilaminoadenosin-3’,5’-monofosforotioato cfclico (Gjertsen et al., 1995); Rp-8-hidroxiadenosin-3’,5’-monofosforotioato cfclico (Gjertsen et al., 1995); los Rp-8-PIP-cAMP (Ogreid et al., 1994); fragmento 14-22 miristoilado inhibidor de PKA (Zhang et al., 2004); fragmento 6-22 de amida inhibidor PKA (Glass et al., 1989); fragmento de amida 5-22 inhibidor de PKA (Cheng et al., 1986); fragmento 5-24 inhibidor de protefna quinasa dependiente de cAMP (Cheng et al., 1986).1998); Rp-8-chloroadenosin-3 ', 5'-cyclic phosphorothioate (Yokozaki et al., 1992); Rp-8- (4-chlorophenylthio) adenosine-3 ’, 5’-cyclic phosphorothioate (Weisskupf et al., 1994); Rp-8-hexylamino-adenosine-3 ’, 5’-cyclic monophosphorothioate (Gjertsen et al., 1995); Rp-8-hydroxyadenosin-3 ’, 5’-cyclic monophosphorothioate (Gjertsen et al., 1995); Rp-8-PIP-cAMP (Ogreid et al., 1994); PKA inhibitor fragment 14-22 PKA (Zhang et al., 2004); PKA inhibitor fragment 6-22 (Glass et al., 1989); PKA inhibitor 5-22 fragment (Cheng et al., 1986); fragment 5-24 cAMP-dependent protein kinase inhibitor (Cheng et al., 1986).
Dentro de la capacidad de la persona experta entra la determinacion de la concentracion optima del inhibidor de PKA en la mezcla de reaccion final.The determination of the optimal concentration of the PKA inhibitor in the final reaction mixture falls within the capacity of the skilled person.
En una realizacion con el inhibidor de PKA es el Rp-adenosin 3’-5’ monofosforotioato cfclico, que se puede adquirir en Sigma-Aldrich con la referencia A165.In one embodiment with the PKA inhibitor it is the Rp-adenosine 3’-5 ’cyclic monophosphorothioate, which can be purchased from Sigma-Aldrich under the reference A165.
Por lo general, el Rp-adenosin 3’-5’ monofosforotioato cfclico se puede usar a una concentracion final de 10 pM.Generally, Rp-adenosine 3’-5 ’cyclic monophosphorothioate can be used at a final concentration of 10 pM.
De acuerdo con otra realizacion de la invencion, el inhibidor de quinasa es un inhibidor de quinasa no selectivo. Algunos inhibidores de quinasa no selectivos adecuados incluyen, pero no se limitan a, H89 (Murray, 2008); KT5720 (Murray, 2008); queleritrina (Freemerman et al., 1996); H7 (Qiu et al., 2010); H8 (Hidaka et al., 1984); inhibidor de protefna quinasa de corazon porcino.According to another embodiment of the invention, the kinase inhibitor is a non-selective kinase inhibitor. Some suitable non-selective kinase inhibitors include, but are not limited to, H89 (Murray, 2008); KT5720 (Murray, 2008); cheleritrine (Freemerman et al., 1996); H7 (Qiu et al., 2010); H8 (Hidaka et al., 1984); porcine heart protein kinase inhibitor.
Por lo general, el inhibidor de quinasa no selectivo puede ser H89, que se puede adquirir en Sigma-Aldrich con la referencia B1427.In general, the non-selective kinase inhibitor can be H89, which can be purchased from Sigma-Aldrich under reference B1427.
H89 se puede usar a una concentracion final de 20 pM.H89 can be used at a final concentration of 20 pM.
Ademas, en otra realizacion, la mezcla de reaccion de ensayo comprende varios inhibidores de quinasa.In addition, in another embodiment, the test reaction mixture comprises several kinase inhibitors.
Despues de realizar la desfosforilacion del sustrato por la calcineurina presente en la muestra biologica, la cantidad de sustrato desfosforilado se cuantifica, ya sea de forma directa o indirecta. Los resultados se pueden expresar como una medida absoluta (tal como pmol/mg de protefna/min), o en unidades arbitrarias.After performing the dephosphorylation of the substrate by the calcineurin present in the biological sample, the amount of dephosphorylated substrate is quantified, either directly or indirectly. The results can be expressed as an absolute measure (such as pmol / mg of protein / min), or in arbitrary units.
Esta etapa de "medir la cantidad de sustrato desfosforilado" se puede realizar ya sea de forma indirecta (por medida de la cantidad de fosfato libre liberado por la reaccion) o de forma directa (por medida del sustrato desfosforilado en sf mismo).This step of "measuring the amount of dephosphorylated substrate" can be performed either indirectly (by measurement of the amount of free phosphate released by the reaction) or directly (by measurement of the dephosphorylated substrate itself).
Se puede usar cualquier metodo para medir la cantidad de sustrato desfosforilado.Any method can be used to measure the amount of dephosphorylated substrate.
Algunos metodos adecuados incluyen, pero no se limitan a:Some suitable methods include, but are not limited to:
- medidas de radiometrfa en el caso de un sustrato etiquetado con 32P (Fruman et al., 1996; Koefoed-Nielsen et al., 2004);- radiometry measurements in the case of a substrate labeled with 32P (Fruman et al., 1996; Koefoed-Nielsen et al., 2004);
- medidas de fluorescencia en el caso de un sustrato etiquetado con un fluoroforo (Roberts et al., 2008);- fluorescence measurements in the case of a substrate labeled with a fluorophore (Roberts et al., 2008);
- medidas de espectrofotometrfa en el caso de sustrato sin etiquetar (Sellar et al., 2006).- spectrophotometry measurements in the case of unlabeled substrate (Sellar et al., 2006).
En una realizacion, la cantidad de fosfato libre se mide usando un ensayo de colorimetrfa y espectrofotometrfa (Sellar et al., 2006). Por ejemplo, el kit de ensayo de actividad celular de calcineurina comercializado por Calbiochem con el numero de catalogo 207007 depende de la deteccion de fosfato libre basandose en el ensayo de verde de malaquita.In one embodiment, the amount of free phosphate is measured using a colorimetry and spectrophotometry assay (Sellar et al., 2006). For example, the calcineurin cell activity assay kit marketed by Calbiochem under catalog number 207007 depends on the detection of free phosphate based on the malachite green assay.
En las tecnicas de deteccion descritas anteriormente, es necesario separar de forma ffsica:In the detection techniques described above, it is necessary to physically separate:
- el fosfato libre del sustrato fosforilado sin metabolizar, por ejemplo, con columnas de cromatograffa con resina de intercambio cationico Dowex (vease Fruman et al., 1996);- phosphorylated substrate free phosphate without metabolizing, for example, with chromatography columns with Dowex cation exchange resin (see Fruman et al., 1996);
- o el peptido desfosforilado del sustrato fosforilado sin metabolizar, por ejemplo usando resinas de oxido de- or the dephosphorylated peptide of the phosphorylated substrate without metabolizing, for example using oxide resins of
titanio (vease Roberts et al., 2008) o usando cromatograffa lfquida (vease Enz et al., 1994) antes de la etapa detitanium (see Roberts et al., 2008) or using liquid chromatography (see Enz et al., 1994) before the stage of
medida en sf misma.measure itself.
En otra realizacion, la cantidad de sustrato desfosforilado se mide mediante cromatograffa lfquida. Algunos metodos de cromatograffa adecuados se describen por ejemplo en Enz et al. (1994).In another embodiment, the amount of dephosphorylated substrate is measured by liquid chromatography. Some suitable chromatography methods are described for example in Enz et al. (1994).
El presente documento describe un metodo para cuantificar de forma simultanea peptidos fosforilados y no fosforilados mediante HPLC acoplada a deteccion de UV. Los experimentos desvelados en el presente documento se realizaron usando calcineurina purificada. Sanquer et al., (2004) tambien describen un metodo de deteccion basado en HPLC acoplada a deteccion de UV para medir la actividad de calcineurina en muestras de PBMC obtenidas de pacientes de trasplante.This document describes a method to quantify phosphorylated and non-phosphorylated peptides simultaneously by HPLC coupled to UV detection. The experiments disclosed herein were performed using purified calcineurin. Sanquer et al., (2004) also describe an HPLC-based detection method coupled to UV detection to measure calcineurin activity in PBMC samples obtained from transplant patients.
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Ademas, en otra realizacion, la cantidad de sustrato desfosforilado se mide mediante cromatograffa liquida acoplada con espectrometrfa de masas en tandem (LC-MS/MS). De forma ventajosa, este metodo de deteccion aumenta adicionalmente la sensibilidad y la especificidad del ensayo.In addition, in another embodiment, the amount of dephosphorylated substrate is measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). Advantageously, this detection method further increases the sensitivity and specificity of the assay.
En una realizacion preferente, la etapa de medir la cantidad de sustrato desfosforilado se realiza mediante cromatograffa liquida acoplada con espectrometrfa de masas en tandem (LC-MS/MS) como se describe en los Ejemplos que siguen a continuacion.In a preferred embodiment, the step of measuring the amount of dephosphorylated substrate is performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) as described in the Examples that follow.
Como se usa en el presente documento, la expresion "muestra biologica" se refiere a cualquier muestra biologica de la que se sospecha que contiene actividad de calcineurina.As used herein, the term "biological sample" refers to any biological sample suspected of containing calcineurin activity.
Por lo general, la muestra biologica puede ser una muestra de sangre, una muestra de celulas mononucleares de sangre periferica (PBMC), una muestra de fluido cerebroespinal o extractos celulares preparados a partir de cultivos celulares.Generally, the biological sample can be a blood sample, a peripheral blood mononuclear cell (PBMC) sample, a cerebrospinal fluid sample or cell extracts prepared from cell cultures.
En una realizacion preferente, la muestra biologica es una muestra de PBMC.In a preferred embodiment, the biological sample is a PBMC sample.
La muestra biologica de acuerdo con la invencion se puede obtener en el momento o se puede mantener a +4 °C o +20 °C antes del analisis.The biological sample according to the invention can be obtained at the time or it can be kept at +4 ° C or +20 ° C before analysis.
En una realizacion preferente, la muestra biologica se usa en un maximo de 7 dfas desde su recogida, preferentemente en 4, 3, 2 o 1 dfas desde la recogida cuando se mantiene a +4 °C o en el dfa de la recogida cuando se mantiene a +20 °C.In a preferred embodiment, the biological sample is used in a maximum of 7 days from its collection, preferably in 4, 3, 2 or 1 days from the collection when kept at +4 ° C or on the day of collection when kept at +20 ° C.
Por lo general, un "paciente" se refiere a un mamffero, tal como un roedor, un felino, un canino, y un primate. Preferentemente, un paciente de acuerdo con la invencion es un ser humano.Usually, a "patient" refers to a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human being.
Por lo general, un paciente de acuerdo con la invencion es un paciente inmunosuprimido, tal como un paciente que ha recibido un trasplante de organos o celulas madre.Generally, a patient according to the invention is an immunosuppressed patient, such as a patient who has received an organ or stem cell transplant.
La presente invencion tambien se refiere a un kit para medir la actividad de calcineurina en una muestra biologica que comprende un sustrato, al menos un inhibidor de fosfatasa y al menos un inhibidor de quinasa.The present invention also relates to a kit for measuring calcineurin activity in a biological sample comprising a substrate, at least one phosphatase inhibitor and at least one kinase inhibitor.
En el kit de acuerdo con la invencion, el sustrato, el inhibidor de fosfatasa y el inhibidor de quinasa son como se han descrito anteriormente.In the kit according to the invention, the substrate, the phosphatase inhibitor and the kinase inhibitor are as described above.
El kit de acuerdo con la invencion puede comprender adicionalmente:The kit according to the invention may further comprise:
- calcineurina recombinante, a usar como control positivo;- recombinant calcineurin, to be used as a positive control;
- controles de calidad- quality controls
- calibradores- calipers
- tampon de ensayo- assay buffer
- un folleto que explica que la reaccion se deberfa realizar en presencia de al menos un inhibidor de quinasa para resultados optimos;- a brochure explaining that the reaction should be carried out in the presence of at least one kinase inhibitor for optimal results;
- y/o medios para detectar la cantidad de fosfato libre liberado por la reaccion, tal como verde de malaquita (para deteccion mediante espectrofotometrfa).- and / or means to detect the amount of free phosphate released by the reaction, such as malachite green (for detection by spectrophotometry).
- y/o un patron interno (para deteccion mediante cromatograffa liquida acoplada a espectrometrfa de masas).- and / or an internal standard (for detection by liquid chromatography coupled to mass spectrometry).
Otro aspecto de la invencion se refiere al uso de inhibidor de quinasa en un metodo para medir la actividad de calcineurina.Another aspect of the invention relates to the use of kinase inhibitor in a method for measuring calcineurin activity.
El metodo para cuantificar la actividad de calcineurina en una muestra biologica tiene muchas aplicaciones.The method for quantifying calcineurin activity in a biological sample has many applications.
Por ejemplo, el metodo se puede usar para controlar la actividad de calcineurina en una muestra biologica obtenida de un paciente al que se le ha administrado un tratamiento inmunosupresor.For example, the method can be used to control calcineurin activity in a biological sample obtained from a patient who has been given an immunosuppressive treatment.
Por lo general, a los pacientes que reciben un trasplante de organos se les proporcionan tratamientos inmunosupresores tales como tacrolimus o ciclosporina.In general, patients receiving an organ transplant are given immunosuppressive treatments such as tacrolimus or cyclosporine.
Por lo tanto, la invencion tambien se refiere a un metodo para predecir el resultado terapeutico de un paciente de trasplante que comprende medir la actividad de calcineurina en una muestra biologica obtenida de dicho paciente como se ha descrito anteriormente.Therefore, the invention also relates to a method for predicting the therapeutic outcome of a transplant patient that comprises measuring calcineurin activity in a biological sample obtained from said patient as described above.
Como se usa en el presente documento, la expresion "predecir el resultado terapeutico" incluye tanto controlar el nivel de actividad de calcineurina en una muestra biologica de un paciente dado en el tiempo (en una primera y segunda muestras biologicas obtenidas en dos puntos temporales distintos) como comparar el nivel de actividad deAs used herein, the expression "predict the therapeutic outcome" includes both controlling the level of calcineurin activity in a biological sample of a given patient over time (in a first and second biological samples obtained at two different time points ) how to compare the activity level of
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calcineurina en una muestra biologica obtenida de dicho paciente con un valor de referencia. En una realizacion, el valor de referencia puede ser un valor obtenido a partir de una poblacion que presenta una enfermedad similar para dicho paciente. Como alternativa, el valor de referencia puede ser un valor obtenido a partir de una poblacion sana.calcineurin in a biological sample obtained from said patient with a reference value. In one embodiment, the reference value may be a value obtained from a population that presents a similar disease for said patient. Alternatively, the reference value may be a value obtained from a healthy population.
Descripcion de las figurasDescription of the figures
Figura 1: Se realizaron experimented cineticos de calcineurina en presencia o ausencia de dos inhibidores de quinasa diferentes. La produccion del peptido DP-RII se cuantifico usando analisis de LC-MS/MS.Figure 1: calcineurin kinetic experiments were performed in the presence or absence of two different kinase inhibitors. DP-RII peptide production was quantified using LC-MS / MS analysis.
Figura 2: Seguimiento longitudinal de actividades de calcineurina y quinasa en 2 pacientes de trasplante. A, B: Paciente 1; C, D: Paciente 2; A, C: Influencia de H89 en la actividad de calcineurina; B, D: la actividad de quinasa se cuantifico por medio de LC-MS/MS.Figure 2: Longitudinal monitoring of calcineurin and kinase activities in 2 transplant patients. A, B: Patient 1; C, D: Patient 2; A, C: Influence of H89 on calcineurin activity; B, D: the kinase activity was quantified by means of LC-MS / MS.
Figura 3: Perfiles cromatograficos de peptido P-RII, peptido DP-RII y peptido RII de isotopo estable en extracto de protefna de PBMC. A, B, C: 1 mg/l de peptido RII en extracted de protefna de PBMC; D, E, F: 1 mg/l de P-RII en extracted de protefna de PBMC; G, H, I: 1 mg/l de peptido RII de isotopo estable en extractos de protefna de PBMC; J, K, L: blanco de extractos de protefna de PBmC; A, D, G, J: transicion 732,4 > 70,1 (peptido P-RII); B, E, H, K: transicion 705,8 > 70,1 (peptido DP-RII); C, F, I, L: transicion 708,3 > 70,1 (peptido RII de isotopo estable).Figure 3: Chromatographic profiles of peptide P-RII, DP-RII peptide and stable isotope RII peptide in PBMC protein extract. A, B, C: 1 mg / l RII peptide in PBMC protein extract; D, E, F: 1 mg / l of P-RII in protein extracted from PBMC; G, H, I: 1 mg / l of stable isotope RII peptide in PBMC protein extracts; J, K, L: blank of PBmC protein extracts; A, D, G, J: transition 732.4> 70.1 (peptide P-RII); B, E, H, K: transition 705.8> 70.1 (DP-RII peptide); C, F, I, L: transition 708.3> 70.1 (stable isotope RII peptide).
Figura 4: Perfiles de supresion de iones para fusion postcolumna de extractos de protefna de PBMC.Figure 4: Ion suppression profiles for postcolumn fusion of PBMC protein extracts.
Figura 5: Reproducibilidad para calibraciones durante un periodo de 12 dfas.Figure 5: Reproducibility for calibrations over a period of 12 days.
EjemplosExamples
Materiales y metodosMaterials and methods
Preparacion de extractos celularesPreparation of cell extracts
Las PBMC obtenidas de voluntarios sanos o de pacientes de trasplante se resuspendieron en un tampon que contenfa Tris 50 mM, pH = 7,0, EGTA 0,1 mM, ditiotreitol 0,05 mM, Tween 20 al 0,1 %, 0,3 mg/ml de albumina, MnCl2 1 mM, CaCh 1 mM e inhibidores de proteasa. Estos extractos celulares se sonicaron en hielo, y se centrifugaron durante 30 minutos a 10.000 g a 4 °C. Los sobrenadantes que corresponden principalmente a las fracciones citosolicas se recuperaron y su contenido de protefna se determino. Estos extractos celulares se pueden almacenar a -80 °C para medida posterior de actividad de CN.PBMCs obtained from healthy volunteers or from transplant patients were resuspended in a buffer containing 50 mM Tris, pH = 7.0, 0.1 mM EGTA, 0.05 mM dithiothreitol, 0.1% Tween 20, 0, 3 mg / ml albumin, 1 mM MnCl2, 1 mM CaCh and protease inhibitors. These cell extracts were sonicated on ice, and centrifuged for 30 minutes at 10,000 g at 4 ° C. The supernatants that correspond mainly to the cytosolic fractions were recovered and their protein content was determined. These cell extracts can be stored at -80 ° C for later measurement of CN activity.
Preparacion de peptidosPeptide Preparation
Se prepararon soluciones de reserva del peptido RII, su forma fosforilada y patron interno (peptido RII de isotopo estable) a una concentracion de 0,5 g/l en tampon Hepes y se almacenaron a -40 °C. Se prepararon calibradores por dilucion de las soluciones de reserva en Tris-HCl para proporcionar un intervalo de calibracion final de 0,16, 0,32, 0,63, 0,95, 1,58, 4,74 y 7,9 pM.Stock solutions of the RII peptide, its phosphorylated form and internal standard (stable isotope RII peptide) were prepared at a concentration of 0.5 g / l in Hepes buffer and stored at -40 ° C. Calibrators were prepared by dilution of the stock solutions in Tris-HCl to provide a final calibration range of 0.16, 0.32, 0.63, 0.95, 1.58, 4.74 and 7.9 pM .
Ensayo enzimaticoEnzymatic assay
De uno a 5 pg de protefnas obtenidas a partir de PBMC, preparadas como se ha descrito anteriormente, se usaron para medir la actividad de calcineurina en una mezcla de reaccion que contenfa acido okadaico 500 nM, 1 mg/l de patron interno, H89 20 pM, 8 mg/l de peptido RII fosforilado y 0,03 unidades de calmodulina en 15 pl de Tris-HCl 50 mM, pH = 7,0. La reaccion enzimatica se realizo a 37 °C durante 30 min y a continuacion, los tubos de reaccion se enfriaron en hielo y la reaccion enzimatica se detuvo mediante la adicion de 15 pl de una solucion de acido tricloroacetico al 5 %. Despues de centrifugacion durante 5 minutos a 12.000 g a 4 °C, los sobrenadantes se transfirieron a inserted de vidrio de 200 pl en viales para autoinyector de vidrio de 1,5 ml y se mantuvieron a 4 °C en automuestreador antes de inyeccion.One to 5 pg of proteins obtained from PBMC, prepared as described above, were used to measure calcineurin activity in a reaction mixture containing 500 nM okadaic acid, 1 mg / l internal standard, H89 20 pM, 8 mg / l phosphorylated RII peptide and 0.03 units of calmodulin in 15 pl of 50 mM Tris-HCl, pH = 7.0. The enzymatic reaction was carried out at 37 ° C for 30 min and then, the reaction tubes were cooled on ice and the enzymatic reaction was stopped by adding 15 pl of a 5% trichloroacetic acid solution. After centrifugation for 5 minutes at 12,000 g at 4 ° C, the supernatants were transferred to 200 pl glass inserted in vials for 1.5 ml glass autoinjector and kept at 4 ° C in autosampler before injection.
Condiciones cromatograficasChromatographic conditions
La LC-MS/MS se realizo usando un sistema de HPLC de la serie 1100 de Agilent conectado a un espectrometro de masas cuadrupolo en tandem API3000 de Applied Biosystems, manejado en el modo de ion positivo con electronebulizacion con control de reaccion seleccionado (sRm). De forma rutinaria se uso una inyeccion de 24 pl, y la cromatograffa se consiguio con una columna Zorbax 300 Extend-C18 de 4,6 x 100 mm y un tamano de partfcula de 3,5 pm, columna analftica protectora de 4,6 x 12,5 mm de Agilent. El caudal era de 0,2 ml/min. La fase movil A era acetato amonico 5 mM y acido formico al 0,1 % en agua y la fase movil B, acetato amonico 5 mM y acido formico al 0,1 % en acetonitrilo. Un gradiente de fase movil programado se uso durante la realizacion de 20 min: 0 min, 2 % de B; 10 min, 100 % de B, 15 min, 100 % de B; 15,5 min 2 % de B, 20 min, 2 % de B. Se seleccionaron peptidos ionicos di-cargados tanto para P-RII, DP-RII como patron interno como los peptidos ionizados de forma mas intensaThe LC-MS / MS was performed using an Agilent 1100 series HPLC system connected to an API3000 tandem quadrupole mass spectrometer from Applied Biosystems, operated in the positive ion mode with electrospray with selected reaction control (sRm) . A 24 pl injection was routinely used, and chromatography was achieved with a 4.6 x 100 mm Zorbax 300 Extend-C18 column and a 3.5 pm particle size, 4.6 x protective analytical column 12.5 mm Agilent. The flow rate was 0.2 ml / min. The mobile phase A was 5 mM ammonium acetate and 0.1% formic acid in water and the mobile phase B, 5 mM ammonium acetate and 0.1% formic acid in acetonitrile. A programmed mobile phase gradient was used during the 20 min realization: 0 min, 2% B; 10 min, 100% B, 15 min, 100% B; 15.5 min 2% of B, 20 min, 2% of B. Di-charged ionic peptides were selected for both P-RII, DP-RII as internal standard and more intensively ionized peptides
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despues de modo de ionizacion positiva con electronebulizacion. El instrumento de MS/MS y los parametros de recogida de datos se muestran en la Tabla 1.after positive ionization mode with electrospray. The MS / MS instrument and the data collection parameters are shown in Table 1.
Tabla 1a: Parametros del instrumento de MS/MSTable 1a: MS / MS instrument parameters
- Tension de pulverizacion de iones (V) Ion Spray Tension (V)
- 5500 5500
- Potencial de entrada Input potential
- 10 10
- Temperatura ( °C) Temperature (° C)
- 50 fifty
- Cortina de gas Gas curtain
- 10 10
- Gas nebulizador Gas nebulizer
- 8 8
- Gas de colision Collision gas
- 4 4
- Resolucion de Q1 Q1 Resolution
- unidad unity
- Resolucion de Q3 Q3 Resolution
- unidad unity
- Tiempo de residencia (ms) Residence time (ms)
- 300 300
- Tipo de barrido Type of scan
- SRM SRM
- Tension de pulverizacion de iones (V) Ion Spray Tension (V)
- 5500 5500
- Potencial de entrada Input potential
- 10 10
- Temperatura ( °C) Temperature (° C)
- 50 fifty
- Cortina de gas Gas curtain
- 10 10
- Gas nebulizador Gas nebulizer
- 8 8
- Gas de colision Collision gas
- 4 4
- Resolucion de Q1 Q1 Resolution
- unidad unity
- Resolucion de Q3 Q3 Resolution
- unidad unity
- Tiempo de residencia (ms) Residence time (ms)
- 300 300
- Tipo de barrido Type of scan
- SRM SRM
Tabla 1.b: Parametros de transicionesTable 1.b: Transition parameters
- compuesto compound
- m/z Q1 m/z Q3 CE CXP DP FP tiempo de retencion m / z Q1 m / z Q3 CE CXP DP FP retention time
- peptido P-RII P-RII peptide
- 732,4 201,2 70 10 100 400 14,5 732.4 201.2 70 10 100 400 14.5
- (sustrato) (substratum)
- 732,4 70,1 90 10 46 320 732.4 70.1 90 10 46 320
- peptido DP-RII DP-RII peptide
- 705,8 70,1 95 8 51 370 14,6 705.8 70.1 95 8 51 370 14.6
- (producto) (product)
- 705,8 201,2 59 20 51 370 705.8 201.2 59 20 51 370
- patron interno internal pattern
- 708,5 70,1 89 8 96 370 14,6 708.5 70.1 89 8 96 370 14.6
- 708,5 201,2 55 18 96 370 708.5 201.2 55 18 96 370
CE: energfa de colision (eV) CXP: potencial de salida de colision celular (eV); DP: potencial de desagregacion (eV) FP: potencial de enfoque (eV).CE: collision energy (eV) CXP: cell collision output potential (eV); DP: disaggregation potential (eV) FP: focusing potential (eV).
Ensayo de validacionValidation test
Los perfiles cromatograficos para blanco de extractos de protefna de PBMC y extractos de protefna de PBMC que contenfan cualquiera de peptido P-RII, peptido DP-RII o patron interno se muestran en la Figura 3. Como se puede observar en la Figura 3, las transiciones seleccionadas para cada peptido de interes son especfficas de un peptido dado, y ninguna de ellas se encuentra en el blanco de extractos de protefna de PBMC. El peptido RII fosforilado presentaba un tiempo de retencion de 14,5 min y tanto el peptido DP-RII como el patron interno presentaban tiempos de retencion similares de 14,6 min.Blank chromatographic profiles of PBMC protein extracts and PBMC protein extracts containing either P-RII peptide, DP-RII peptide or internal standard are shown in Figure 3. As can be seen in Figure 3, the Selected transitions for each peptide of interest are specific to a given peptide, and none of them is on the target of PBMC protein extracts. The phosphorylated RII peptide had a retention time of 14.5 min and both the DP-RII peptide and the internal standard had similar retention times of 14.6 min.
En la Figura 4 se muestran algunos perfiles representativos de supresion de iones para un extracto de protefna de PBMC analizado por LC-MS/MS 4. Los efectos de supresion se produjeron principalmente entre 6 y 11 minutos de la cromatograffa, a continuacion la senal se estabilizo de forma gradual. El pequeno efecto de supresion de iones observado en los tiempos de elucion de los peptidos de interes se puede ignorar debido a la coelucion del patron interno.Some representative profiles of ion suppression for a PBMC protein extract analyzed by LC-MS / MS 4 are shown in Figure 4. The suppression effects occurred mainly between 6 and 11 minutes of chromatography, then the signal was I stabilize gradually. The small effect of ion suppression observed in the elution times of the peptides of interest can be ignored due to the coelution of the internal pattern.
La reproducibilidad del ensayo se verifico en 7 calibraciones separadas realizadas durante un periodo de 12 dfas como se muestra en la Figura 5. Cada calibracion individual presentada un R2 de 0,9966 o superior. La ecuacion de regresion media para las 7 calibraciones, en las que el coeficiente de regresion se expresa como media (DT), eraThe reproducibility of the assay was verified in 7 separate calibrations performed over a period of 12 days as shown in Figure 5. Each individual calibration presented an R2 of 0.9966 or higher. The mean regression equation for the 7 calibrations, in which the regression coefficient is expressed as mean (SD), was
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como sigue a continuacion: y = [0,1863 (0,055)]x-[0,0007 (0,00421)].as follows: y = [0.1863 (0.055)] x- [0.0007 (0.00421)].
La precision del ensayo se examino en extractos de protema de PBMC con actividades de calcineurina conocidas que variaban de 90 a 500 pmol/mg/min. La variabilidad maxima intraensayo e interensayo era de un 11,5 % y un 16 %, respectivamente (Tabla 2).The accuracy of the assay was examined in PBMC protein extracts with known calcineurin activities ranging from 90 to 500 pmol / mg / min. The maximum intra-assay and inter-assay variability was 11.5% and 16%, respectively (Table 2).
Tabla 2: Datos de imprecision para actividad de calcineurina medidos en extractos de protefna de PBMCTable 2: Inaccuracy data for calcineurin activity measured in PBMC protein extracts
- Intraensayo Intraassay
- Interensayo Intensay
- nombre Name
- media medida nombre media medida half measure name half measure
- de la muestra of the sample
- pmol/mg/min CV, % de la muestra pmol/mg/min CV, % pmol / mg / min CV,% of sample pmol / mg / min CV,%
- 1 one
- 92 6,6 7 337 10,5 92 6.6 7 337 10.5
- 2 2
- 232 4,6 8 91 8,7 232 4.6 8 91 8.7
- 3 3
- 333 10,6 9 160 11,5 333 10.6 9 160 11.5
- 4 4
- 132 8,6 10 375 8,0 132 8.6 10 375 8.0
- 5 5
- 221 11,5 11 362 5,6 221 11.5 11 362 5.6
- 6 6
- 116 4,3 12 454 13,5 116 4.3 12 454 13.5
- 13 452 11,7 13 452 11.7
- 14 356 12,8 14 356 12.8
- 15 140 12,3 15 140 12.3
- 16 179 16,0 16 179 16.0
ResultadosResults
Ninguno de los metodos descritos anteriormente analizaba la posibilidad de refosforilacion del peptido DP-RII durante el proceso de reaccion enzimatica de CN con quinasas presentes en los extractos celulares.None of the methods described above analyzed the possibility of refosphorylation of the DP-RII peptide during the enzymatic reaction of CN with kinases present in cell extracts.
Los inventores evaluaron la cinetica de la calcineurina en presencia o no de 2 inhibidores de quinasa diferentes: H89, un inhibidor inespedfico y Rp-adenosin 3',5'-monofosforotioato dclico, un inhibidor espedfico de PKA.The inventors evaluated calcineurin kinetics in the presence or absence of 2 different kinase inhibitors: H89, an unspecified inhibitor and Rp-adenosine 3 ', 5'-dichloric monophosphorothioate, a specific PKA inhibitor.
Un primer conjunto de experimentos se realizo usando 5 pg de protemas citosolicas de PBMC obtenidas a partir de voluntarios sanos. Las celulas se resuspendieron en un tampon que contema Tris 50 mM, pH = 7,0, EGTA 0,1 mM, ditiotreitol 0,05 mM, Tween 20 al 0,1 %, 0,3 mg/ml de albumina, MnCh 1 mM, CaCl2 1 mM e inhibidores de proteasa. Las reacciones enzimaticas se procesaron a 37 °C en el tampon Tris 50 mM, pH = 7,0, complementado con 8 mg/l detective RII-P, 0,03 unidades de calmodulina, acido okadaico 500 nM y 1 mg/l peptido RII de isotopo estable como un patron interno. Las reacciones enzimaticas se detuvieron en los puntos temporales indicados (hasta 120 min) y las cantidades de peptido se cuantificaron por LC-MS/MS.A first set of experiments was performed using 5 pg of PBMC cytosolic proteins obtained from healthy volunteers. The cells were resuspended in a buffer containing 50 mM Tris, pH = 7.0, 0.1 mM EGTA, 0.05 mM dithiothreitol, 0.1% Tween 20, 0.3 mg / ml albumin, MnCh 1 mM, 1 mM CaCl2 and protease inhibitors. Enzymatic reactions were processed at 37 ° C in 50 mM Tris buffer, pH = 7.0, supplemented with 8 mg / l RII-P detective, 0.03 units of calmodulin, 500 nM okadaic acid and 1 mg / l peptide Stable isotope RII as an internal standard. Enzymatic reactions were stopped at the indicated time points (up to 120 min) and the peptide amounts were quantified by LC-MS / MS.
Como se muestra en la Figura 1, la produccion de peptido DP-RII era significativamente mas elevada en presencia de los inhibidores de quinasa (inhibidor espedfico de PKA con respecto a control, p = 0,0008; H89 con respecto a control, p = 0,0007, ensayo de ANOVA). Ademas, la inhibicion obtenida con H89 era significativamente mas elevada que la obtenida con el inhibidor espedfico de PKA (p = 0,0163).As shown in Figure 1, the production of DP-RII peptide was significantly higher in the presence of kinase inhibitors (PKA specific inhibitor with respect to control, p = 0.0008; H89 with respect to control, p = 0.0007, ANOVA trial). In addition, the inhibition obtained with H89 was significantly higher than that obtained with the specific PKA inhibitor (p = 0.0163).
Para examinar la importancia de tal influencia en la estrategia farmacodinamica para controlar el alcance de la inmunosupresion despues del trasplante, los inventores evaluaron las PBMC de pacientes de trasplante en presencia o ausencia del inhibidor de quinasa H89, en un segundo conjunto de experimentos. Como se muestra en la Figura 2, una fuerte variacion en la actividad de quinasa se observo entre pacientes y en el tiempo. Ademas, la actividad de quinasa era capaz de alterar la medida de la actividad de CN, lo que refleja la importancia del enfoque de los inventores.To examine the importance of such influence on the pharmacodynamic strategy to control the extent of immunosuppression after transplantation, the inventors evaluated the PBMC of transplant patients in the presence or absence of the H89 kinase inhibitor, in a second set of experiments. As shown in Figure 2, a strong variation in kinase activity was observed between patients and over time. In addition, the kinase activity was able to alter the measure of CN activity, reflecting the importance of the inventors' approach.
Este hallazgo demuestra que la medida de la actividad de CN se puede realizar en presencia de un inhibidor de quinasa, para obtener una medida mas espedfica.This finding demonstrates that the measurement of CN activity can be performed in the presence of a kinase inhibitor, to obtain a more specific measurement.
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| EP12305231 | 2012-02-27 | ||
| PCT/EP2013/052943 WO2013127636A1 (en) | 2012-02-27 | 2013-02-14 | Method for measuring calcineurin activity |
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| CA2694329A1 (en) * | 2007-08-01 | 2009-02-05 | Emory University | Methods for determination of protein phosphatase activity, and uses in predicting therapeutic outcomes |
| CN102712948A (en) * | 2010-01-08 | 2012-10-03 | 爱默蕾大学 | FRET-based method for the determination of protein phosphatase and kinase activity |
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